Chemical supplementation of bone

ABSTRACT

Disclosed and claimed is bone or bone tissue supplemented with a therapeutically effective compound and methods for supplementing bone or bone tissue with the therapeutically effective compound.

The present invention provides bone and bone tissue supplemented with atleast a therapeutically useful compound and in particular relates to amethod for supplementing bone and or bone tissue with said compound(s).

Major allograft surgery has provided a solution to many reconstructiveproblems in musculoskeletal and maxillofacial surgery. The use of suchsurgery remains however, retarded by the frequency of infections thatare often a disabling complication of such surgery. While the use ofsmall frozen allografts has a very low rate of infection, majorallografts have infection rates between 5 and 13%. This susceptibilityto infection is probably multifactorial, with avascularity andantigenicity of the implanted graft contributing as well as the frequentextensive soft tissue excision, and potential for wound breakdown.

The use of allograft bone in orthopaedic practice is now wellestablished both as morsellised and site specific structural grafts. Therisk of infection, however remains a major complicating factor with suchsurgery. Like other forms of allograft surgery, the frequency ofinfection with allograft bone varies between 5 and 13.3%. The outcome inpatients who develop infection is poor and often requires either twostage revision or amputation.

Infections typically arise early after allograft surgery with 75% ofcases presenting within 4 months. Perioperative introduction oforganisms is the presumptive mode of infection in the majority of thesecases. The most common organisms isolated are gram positive (54%)followed by gram negative (36%) and mixed (10%).

Numerous attempts have been made to lessen the rate of infection inallograft surgery, particularly in the field of maxillofacial surgery.Perioperative antibiotic regimes are often employed, involving prolongedadministration of antibiotics for up to 3 months, although no controlledstudies have been performed to show the efficacy of these regimes. Thetheoretical problem of systemic antibiotic administration in allograftsurgery, particularly when using allograft bone is that the allograftsare initially avascular and the antibiotics do not reach their target.

Attempts have been made to load allograft bone with antibiotics. In onesuch study morsellised graft was mixed with antibiotic solutions. Morerecently antibiotic supplemented bone allograft has been developed andused in the area of avulsive defects of the oral and maxillofacialskeleton. This technique employs demineralised particulate allograftbone and mixes it with purified gelatine powder and cephalothin andtobramycin. A canine model to test this preparation has shown a probableprotection from post-operative infection when compared with conventionalallografts.

Although these methods have been shown to display a decreasedcomplication rate, the problem of infection in major allograft bonesurgery is still a major concern. Furthermore, present methods ofpreparing allograft bone against infection require a large amount ofpreparatory work, are typically unsuitable where large bone grafts arerequired and depending on the methods used may not result in a productthat has the same structural integrity as allograft bone. Thus, theproblem of infection in major allograft bone surgery is largely unsolvedand has severe consequences to patients who develop complications.

The present invention seeks to provide an improved bone and or bonetissue supplemented with at least a therapeutically useful compound.Moreover, the invention seeks to provide a simple and effectiveprocedure for supplementing bone and or bone tissue with at least atherapeutically effective compound.

Throughout the specification, unless the context requires otherwise, theword “comprise” or variations such as “comprises” or “comprising”, willbe understood to imply the inclusion of a stated integer or group ofintegers but not the exclusion of any other integer or group ofintegers.

For the purposes of the present invention the phrase “bone and bonetissue” encompasses bone substitutes which comprise any biological orsynthetic material used to substitute for bone during reconstructionincluding material processed from xenograft sources and chemicalsmanufactured for bone substitute purposes such as calcium phosphate andhydroxyappatite.

The present invention consists in a bone or bone tissue supplementedwith at least a therapeutically useful compound, wherein said compoundis concentrated within the bone matrix.

Unlike prior art products the present invention does not rely upon theuse of binders, protective agents, gelatinisation agents or the like toassociate therapeutic compounds with allograft bone or tissue. Rather,therapeutically effective compounds are delivered to and concentratedwith the bone matrix by a process of iontophoresis. Preferably theconcentration of the therapeutically effective compound within the boneis greater than the amount of therapeutically effective compound thatmight be absorbed into bone as a result of simple diffusion. It will beappreciated that the relative amount of therapeutic compound which mightbe loaded into any particular piece of bone will depend on (a) the safein situ usage limits for that therapeutic compound, (b) thecharacteristics of the bone or bone tissue, (c) the biochemicalcharacteristics of the particular compound selected and (d) theparticular purpose for which the bone or bone tissue is being used.

Therapeutically effective compounds that might be employed in theinvention include, but are not limited to: antibiotics, antifungalcompounds and chemotherapeutic compounds, tissue growth factors (forexample bone morphogenic protein), non-steroidal anti-inflammatoryagents, such as indomethacin, neuromuscular agents affecting calcium andbone metabolism (such as calcitonin), anti-viral agents,anti-tuberculosis agents (such as rifampicin), anthelmintic agents (suchas mebendazole), antiseptic agents, vitamins and minerals. Mostpreferably, the compounds that are loaded into the bone are compoundsthat form a salt in solution and ionise to a single positive or negativeion. Those of ordinary skill in the art will know such compounds.

If, for example, antibiotics are to be loaded into the bone or bonetissue the antibiotic compound is preferably selected from thefollowing: flucloxacillin, gentamicin, cephalothin, ticarcillin,ciprofloxacin, nenzl-peniccillin, cefoperazone, cefuroxime, cephazolinand tobramycin. Most preferably the antibiotic is either flucloxacillinor gentamicin. When loaded into bone these compounds are preferablypresent at a concentration of between the minimum inhibitoryconcentration of the antibiotic and the concentration that would providea total amount of antibiotic equal to the safe maximum single dose forsystemic administration. For example, the maximum dose of gentamicinthat might be loaded into allograft bone is about 200 mg/kg while themaximum dose of flucloxacillin is about 80 mg/kg.

If the therapeutically effective compound is an antifungal compound, theantifungal compound is preferably selected from the following:miconazole, and ketaconazole. When loaded into bone, these compounds arepreferably present at a concentration of between the minimum inhibitoryconcentration of the antifungal and the concentration that would providea total amount of antifungal equal to the safe maximum single dose forsystemic administration.

If the therapeutically effective compound is a chemotherapeutic compoundthe chemotherapeutic is preferably selected from the following:5-fluoro-uracil and vinblastin. Most preferably the chemotherapeutic is5-fluoro-uracil.

In an alternative form, the present invention consists of a method forsupplementing bone or bone tissue with a therapeutically effectivecompound, wherein said method employs the steps of:

(i) Exposing bone or bone tissue to a therapeutically effectivecompound; and

(ii) Applying a potential difference across said bone or bone tissuesuch that the therapeutically effective compound is concentrated withinthe bone or bone tissue.

Preferably, the therapeutically effective compound employed in themethod is concentrated within the bone or bone tissue using anexternally applied potential difference. Any externally appliedpotential difference may be used in the method, provided that it doesnot destroy the structural integrity of the bone or bone tissue. Thepotential difference that is used will depend on: (a) the thickness ofthe bone, (b) the time available to deliver the compound to the bone,(c) the compound which is to be loaded into the bone and (d) thetemperature of the bone. Preferably the temperature of the bone duringthe loading process should be maintained below about 37° C.

If highly externally applied potential differences are being used toload the bone with a therapeutically effective compound, then the methodshould be carried out in the presence of a means which is capable ofcooling the bone or bone tissue. For example the method might be carriedout in a refrigerated environment or alternatively might be carried outin a water bath.

It will be appreciated that the present invention is not limited to theloading of sectioned allograft bone. It might also be used in situ todeliver compounds into bone to treat medical disorders such as bonetumours. In such circumstances the therapeutically effective compound ispreferable introduced at medically safe levels into the tissuesurrounding the bone. An externally applied potential difference is thenapplied across the bone for sufficient time to concentrate thetherapeutically effective compound within the bone. Preferably, theexternally applied potential difference is selected such that it iscapable of drawing and concentrating the therapeutically effectivecompound into the bone but does not effect the structural integrity ofthe surrounding tissue.

Therapeutically effective compounds suitable for use in the method arethose which are capable of forming a soluble salt in solution.Preferably the compounds selected are capable of ionising in thepresence of an externally applied potential difference to form eitherpositive or negative ions. Examples of suitable compounds are describedabove. Most preferably antibiotics such as flucloxacillin and gentamicinare used as the therapeutically effective compounds.

The concentration of therapeutically effective compounds that may beloaded into the bone or bone tissue will depend largely on theproperties of the compound used and the time over which the compound isrequired to have a therapeutic effect. Preferably the compound isconcentrated within the bone to a level which exceeds the amount ofcompound that might be diffused into the bone as a result of diffusionover an equivalent period of time. That is, when both the present methodand a diffusion method are carried out over an equivalent period oftime.

Applying an external potential difference across bone or bone tissuerequires the use of at least two electrodes, one being located on oneside of the bone and the other being suitably positioned on the otherside of the bone. To ensure electrical contact between the electrodesthey are each preferably surrounded by a medium capable of conductingelectrical current. Preferably there is a plurality of electrodes oneither side of the bone. While any electrode might be used in themethod, the preferred electrodes are those that do not produce achemical residue that would damage the bone or bone tissue. Suitableelectrodes for use in the invention include, but are not limited to,carbon, platinum, titanium, gold, noble metals, stainless steel,conductive plastic and the like.

In a highly preferred form of the invention the method is applied toallograft bone to load the bone with suitable therapeutic compoundsprior to or during allograft surgery. Preferably the method is performedunder aseptic conditions.

According to a particularly preferred form of the method, the section ofbone to be treated is prepared and defrosted. It is then cut (in amanner which would be well known to those in skilled in the art) to anappropriate length with a slight excess at each end. Preferable thatexcess is in the order of about 1 to 10 mm.

To one end of the bone, a disc of an appropriate size to completely sealthe medullary canal at that end is sealingly engaged to the bone. Thedisc can be made of any insulating material, such as acrylic, plasticetc. Sealing engagement between the bone and the disc may be achievedusing, for example, a glue which is capable of bonding the disc to thebone, such as cyanoacrylate, and which is capable of being sterilised.Those of ordinary skill in the art will know such glues.

To the opposite end of the bone, a tube of an appropriate length iscemented to the bone, such that the tube sealingly engages onto the endthe medullary canal (see FIG. 1). The tube can be made of any insulatingmaterial, such as acrylic. The specimen is then placed in a beaker andimmersed in a buffer solution such that the open end of the extendedmedullary canal is just above the level of the ionic solution. Thebuffer solution can be any solution capable of conducting electricalcurrent, such as normal saline.

The medullary canal is then filled with an ionic solution of thecompound to be loaded into the bone. Desirably the solution is sterileand consists of an antibiotic in ionic form such as gentamicin orflucloxacillin, but may also consist of antifungal compounds,chemotherapeutic agents or any other compound that ionises to a singleionic species in solution. The pH of solution should then be optimisedto ensure maximal ionisation of compound.

Electrodes are then placed in the apparatus, with at least one placedvertically in the ionic solution, and a plurality of vertical, equallyspaced electrodes fixed to the side of the beaker, immersed in thebuffer solution. The surrounding electrodes should then be connectedelectrically such that they act as one electrode. The electrodes can bemade of any inert material such as carbon, or platinum.

A potential difference is then applied across the electrodes untilloading of the therapeutically effective compound into the bone iscomplete. The time required for the compound to be transferred in to thebone tissue will depend on (a) the voltage—with increasing voltage theshorter the time period is required for transfer of the compound, (b)the thickness of the bone (c) the ionic compound used. Typically themaximum voltage that might be used in the method without the assistanceof a suitable cooling means would be in the order of 100 V, althoughthere is theoretically no upper or lower limit to the voltage that canbe used when a cooling means is employed. Desirably the temperature ofthe bone should not reach or exceed 37° C., at which temperature thecollagen component of bone begins to degrade.

Voltages and times for application may be determined experimentally asdescribed in the following examples.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further described by the following non-limitingFigures and Examples.

FIG. 1 illustrates a representative apparatus for transverseiontophoresis.

FIG. 2 illustrates a representative apparatus for longitudinal orcircumferential iontophoresis.

FIG. 3 illustrates a apparatus for iontophoresis of tubular bonespecimens.

FIG. 4 illustrates a graph of gentamicin iontophoresis using sheeptibia.

FIG. 5 illustrates a graph of flucloxacillin iontophoresis using sheeptibia.

FIG. 6 illustrates a graph of gentamicin iontophoresis using humanallograft.

FIG. 7 illustrates a graph of flucloxacillin iontophoresis using humanallograft.

FIG. 8 illustrates a graph of the combined iontophoresis usinggentamicin and flucloxacillin.

FIG. 9 illustrates a graph of hourly gentamicin washout per mgantibiotic available.

FIG. 10 illustrates a graph of hourly flucloxacillin washout per mgantibiotic available.

FIG. 11(a) illustrates gentamicin bioactivity against StaphylococcusAureus.

FIG. 11(b) illustrates flucloxacillin bioactivity against StaphlococcusAureus.

FIG. 12 illustrates a graph of a temperature plot.

Further features of the present invention are more fully described inthe following Examples. It is to be understood, however, that thisdetailed description is included solely for the purposes of exemplifyingthe invention, and should not be understood in any way as a restrictionon the broad description as set out above.

EXAMPLES Example 1

Experimental work was carried out using mature Merino Sheep tibiaeaccessed from ‘butcher's shops’, and later using Human allograft bonewhich was rejected for implantation by the Perth Bone and Tissue Bank.The ionic compound used initially was methylene blue in 1% solutionwhich forms positive ions and migrates away from the anode when apotential difference is applied, flucloxacillin and gentamicin were theantibiotics selected as the antibiotics for further study. Methyleneblue was chosen for the initial work because its ease of visualidentification obviated the need for assay techniques. It has amolecular weight of 373 which is not very different from that offlucloxacillin (453) and gentamicin (460). This similarity means that itprovides a good model to study the likely behaviour patterns offlucloxacillin and gentamicin ions using iontophoresis. Gentamicinexists in solution as positive ions of the sulphate salt, andflucloxacillin as negative ions and ionic aggregates of its sodium salt.

Preliminary work was done using 1 cm² discs or squares of cortical boneand was used to establish whether iontophoresis was effective infacilitating the movement of methylene blue ions in bone. In additionthe technical aspects of how the technique's efficacy could be maximisedwere studied with particular reference to: The direction in whichmethylene blue is moved easiest under the influence of a potentialdifference through cortical bone; The effect of a pulsed field ascompared with a unipolar potential gradient on the movement of ions; andthe effects of varying the potential difference on the rate of movementof ions across the bone.

Pieces of bone were imbedded in epoxy cement, as an electricalinsulator, leaving 2 surfaces free to apply the iontophoretic gradientacross. The specimens were then sectioned and the penetration of themethylene blue measured. The apparatus used is shown in FIGS. 1 & 2.

These served to study the feasibility of iontophoresis in bone, and themost effective method of applying the technique. Control experimentswhere methylene blue was applied to one surface of the cortical bonedisc revealed very minimal penetration of the blue colour even after 20minutes. In cases where a potential difference was applied the methyleneblue was observed to migrate through the cortices.

The rate of facilitated diffusion was proportional to the potentialdifference applied and at 100 volts across a 4 mm cortex, fullpenetration could be expected at approximately 4 minutes. There was noadvantage apparent from using a pulsed field, as polarisation of theelectrodes with a consequent fall in the current being passed appears tooccur at about the time of full cortical penetration, by the ions underinvestigation, and is therefore of little consequence. The technique wasmost effective in moving ions transversely across the cortex. It wasless effective at moving ions circumferentially around a cortex orlongitudinally in the direction of the haversian canals.

Example 2

Tubular Bone Allograft Model:

From this initial work a system to fully investigate the hypothesis wasdeveloped with more clinical relevance; Sections 2 cm long were cut fromthe diaphysial region of the sheep tibia. They were sealed at one end bycementing on an acrylic disc using cyanoacrylate glue. The open end ofthe tube of bone had a 3 cm long acrylic tube cemented to it,effectively extending the medullary canal and thereby increasing itsvolume. The specimen was then placed in a beaker to the inside of which3 vertical equally spaced carbon electrodes were fixed. The beaker wasthen filled with normal saline solution to just below the open end ofthe acrylic tube. Two ml of the ionic compound under investigation wasplaced in the medullary canal of the tibia and a carbon electrodelowered into it. The effect of a potential difference applied across thecortex could thus be studied. Control specimens were prepared in anidentical fashion, except no potential difference was applied across theelectrodes. Study specimens had the potential difference applied for 1minute, 2 minutes, 5 minutes or 10 minutes.

All specimens were then removed and the medullary canal washed clean anddried. Axial sections were then cut using a diamond saw and the amountof penetration of the methylene blue measured. In addition histologicalsections were prepared 300 μm thick to assess the penetration at amicroscopic level. The methylene blue iontophoresis provided aqualitative analysis of iontophoresis in bone.

With the system set up as described, and methylene blue as the ionicsolution the typical current that flowed during iontophoresis of thespecimens was 40 mamp. The blue coloration of the periosteal surface wasnoted by approximately 1.5 minutes and by 5 minutes was maximal.

Macroscopic evaluation of the specimens subsequently was done toevaluate the penetration of the methylene blue and microscopicevaluation done to assess the uniformity of penetration. The results ofthis are shown in Table 1

This work was duplicated using sections of human allograft tibia. Theresults were found to be comparable to the sheep model. The onlyappreciable difference between the two was that the sheep tibiaeconsisted purely of compact cortical bone whereas the human tibiaecomprised cortical bone on the periosteal aspect, with cancellous boneon the endosteal aspect. This cancellous bone was no barrier todiffusion of the methylene blue. The rate of facilitated diffusion andthe uniformity of penetration were comparable.

TABLE 1 Macroscopic Microscopic Control Minimal Penetration (Staining ofStaining on endosteal endosteal Surface with few surface with volkman'sVolkman's canals staining for canals filled for variable variabledistance) distance. No staining of bony matrix 1 Minute Penetration 1.5mm Uniform staining of matrix to level of penetration. Filling ofVolkman's canals through full thickness of cortex 2 Minutes Penetration3 mm Uniform staining of matrix to level of penetration. Filling ofVolkman's canals through full thickness of cortex 5 Minutes Penetrationfull thickness of Complete uniform cortex. (Patchy staining ofperiosteal penetration of entire surface) thickness of cortical bonematrix and Volkman's canals

Example 3

Quantitative analysis was then carried out to test iontophoresis as ameans of antibiotic delivery in allograft bone. Specimens of sheep tibiawere prepared as described above. The medullary canal was filled with 2ml of 1% gentamicin Sulphate solution (Delta West, Western Australia) or1% flucloxacillin Sodium solution (Alphapharm) in distilled water. Apotential difference of 100 volts was applied for 1 minute, 5 minutes or10 minutes with controls being set up the same way for 10 minutes exceptno potential difference was applied. A recording of current passed foreach specimen was made.

Each sample was then washed in water and dried. Samples from 10 theendosteal and periosteal surfaces were then taken by drilling the boneusing a 3 mm low speed drill, in 10 positions on each surface, andcrushed to a fine powder. To a known mass of each sample was addeddistilled water (2 ml) and the sample agitated using ultrasound. Thesample was then centrifuged and the supernatant removed and the processrepeated a further 2 times. The last wash was left to soak for 12 hoursbefore centrifuging. The Fucloxacillin specimens were refrigerated to 4°C. during this to minimise degradation by hydrolysis. The supernatantsolutions were then analysed for antibiotic content; for gentamicinusing Fluorescence Polarisation Immuno Assay (Abbot Axsym Analyser); andfor flucloxacillin the assays were performed using High PerformanceLiquid Chromatography (HPLC) technique. From the levels in thesupernatant solutions, the antibiotic concentrations in the bone sampleswere calculated.

To study the thermal effects of iontophoresis on bone, some samples wereprepared and a fibre optic thermal fluorescence probe was introducedinto the centre of the cortical bone via a 0.8 mm drill hole in the longaxis of the specimen. The apparatus was otherwise set up as above andthen cooled to 10° C. The iontophoretic potential difference was thenapplied at 100 volts for 15 minutes and a continuous recording of thetemperature made.

Initial work was carried out using the sheep tibia model of allografttubular bone. For each control and iontophoretic group, 5 specimens wereused. Samples from the endosteal and periosteal surfaces were processedseparately. The results are shown in FIGS. 4 & 5.

It can be seen from these graphs, that both flucloxacillin andgentamicin ions are moved through sheep cortex by the iontophoreticgradient. The levels of gentamicin achieved in both endosteal andperiosteal specimens plateau at about 150 mg/Kg. This compares withrecommended peak serum concentrations after intravenous administrationof the drug of 10 mg/L. The minimum inhibitory concentration ofgentamicin to Staphylococcus aureus is approximately 0.25 mg/L (althoughthis varies with different phage types). The peak in the endostealspecimens was reached by 1 minute, and the periosteal samples at 5minutes. This is due to the fact that the ions have further to travel toreach the periosteal surface, and consequently take longer.

This work was supplemented by repeating the study using human allografttibia. The scarcity of available allograft bone necessitated that eachgroup contained only one specimen. All 4 specimens were contiguous 1 cmsections of the diaphysis of the same non-irradiated tibia for each ofgentamicin and flucloxacillin. The results are shown in FIGS. 6 & 7.

Gentamicin and flucloxacillin behave in a similar fashion in humanallograft bone to sheep bone. Again therapeutic levels of antibiotic areachieved although the time to reach maximal levels at all depths was 10minutes. This increase in time probably reflects the increase thicknessof the human specimens compared with the sheep specimens.

As gentamicin migrates from the anode and flucloxacillin migrates fromthe cathode, the next step to investigate was the possibility ofsimultaneous iontophoresis of each drug in opposite directions acrosssheep tibia specimens. The set up was as previously used, the gentamicin1% solution being placed in the medullary canal with the anode and theflucloxacillin 1% solution being placed in the beaker, thus bathing thecathode. The results of this are shown in FIG. 8.

As can be seen here, gentamicin penetrates the full thickness of thecortex after 5 minutes of iontophoresis, whereas the flucloxacillinlevels in the periosteal samples only are elevated. The total antibioticconcentration is markedly elevated when compared with the controls.

Example 4

The bioavailability of the antibiotics was assessed by creatingspecimens using the above iontophoretic technique and applying thepotential difference for 5 minutes. Samples were then drilled from thecortices and prepared as above for assay of the starting antibioticconcentration. Each specimen was then immersed in 20 mls of NormalSaline. This solution was poured off at intervals and antibiotic levelsassayed in the solution. Each sample was immediately re immersed infresh solution. In this way the rate of elution of the iontophoresedantibiotic could be studied. Again, the gentamicin samples were kept atambient temperature the flucloxacillin samples being refrigerated to 4°C. The final elution assay was carried out at 14 days. The bioactivityof the antibiotics loaded into bone by iontophoresis was investigated.Specimens of sheep tibia were prepared: ⅓ having had no exposure toantibiotics, ⅓ having been soaked for 5 minutes in antibiotic solutionand ⅓ having been loaded with antibiotic using iontophoresis as per theabove technique for 5 minutes with a potential difference of 100 volts.From each specimen a 1 mm thick axial section was cut using a diamondsaw and this was placed on a nutrient agar plate with a standardStaphylococcus aureus preparation (fully sensitive). The plates werethen incubated for 24 hours and the zone of inhibition inspected andmeasured.

The results for the rates of dissolution of the antibiotics from tubularsections of bone 2 cm long, (approx. 5 g weight) in 20 ml of saline areshown in FIGS. 9 & 10.

Both antibiotics behaved in a similar fashion, with a logarithmicdecrease in their rates of dissolution. There was still antibioticbecoming available up to two weeks after soaking the bone in normalsaline solution.

Bioactivity of Antibiotics after Iontophoresis: The results of thenutrient agar plate test of the drugs activity against a fully sensitiveStaphylococcus are shown in FIGS. 11a and 11 b.

While bacterial colonies can be seen growing up to the bone in the caseof bone which has never been exposed to antibiotic there is a zone ofinhibition of growth around the bone for those soaked in antibiotic anda larger zone of inhibition around the iontophoresis specimens.

Thermal Effects of Iontophoresis: The temperature plot is shown in FIG.12 for the mean values of 3 specimens. All specimens were initiallycooled to 10° C. The plot shows the mean values with the highest andlowest values superimposed. The iontophoretic current was turned off at15 minutes and recordings of temperature made for a further 3 minutes.

After an initial rapid rise in the first 30 seconds the temperatureincreases slowly until the current is turned off at 15 minutes. Thehighest temperature recorded was 35.7° C. This thermal effect is resultof the energy dissipation of the 40 mamp current being passed at 100volts potential difference representing a power generation of 4 wattsenergy, within the system as a whole.

The results of this work demonstrate that high concentrations of bothgentamicin and flucloxacillin can be achieved in sheep tibia after 1minute and human allograft tibia after 5 minutes of iontophoresis. Theprocedure is simple and requires no expensive equipment. Furtherallograft bone may be treated in this way at the time of defrosting thegraft prior to implantation in the operating room.

An attempt to simultaneously move gentamicin and flucloxacillin inopposite directions across a cortex has shown that gentamicin moves fullthickness, but flucloxacillin penetrates only the periosteal side. Thisis most likely due to the precipitation reaction encountered whensolutions of gentamicin and flucloxacillin are mixed. The implication inclinical practice is that the gentamicin will penetrate well using thissystem, but the periosteal surface of the bone will in addition achievetherapeutic levels of flucloxacillin. The preliminary work withmethylene blue has shown that this is likely to be the surface thatreceives the least predictable amount of positively charged ion (in thiscase, gentamicin.).

While the levels of gentamicin in the bone may appear alarmingly high,if a 500 g allograft containing 200 mg/Kg were to be implanted, thetotal dose of drug being implanted is about 100 mg, which is well belowthe usual daily dose of gentamicin on a once daily intravenous regime.The method of calculation of antibiotic concentration in bone hasassumed that all antibiotic is washed out of the crushed bone. Thelevels of antibiotic described above are also considerably higher thanlevels that have been measured in bone after intravenous administrationwhich vary from 2.4-19.4 mg/Kg and usually reach levels between 0.14 and0.36 of serum concentration.

It should be understood that the foregoing description of the inventionincluding the principles, preferred embodiments and Examples cited aboveare illustrative of the invention and should not be regarded as beingrestrictive on its scope. Variations and modifications may be made tothe invention by others without departing from the spirit of that whichis described as the invention and it is expressly intended that all suchvariations and changes which fall within this ambit are embraced therebyis intended merely to be illustrative thereof.

The claims defining the invention are as follows:
 1. A method forsupplementing bone or bone tissue with a therapeutically usefulcompound, comprising the steps of: (a) exposing bone or bone tissue invitro or ex vivo to a therapeutically useful compound; and (b) applyinga potential difference across said bone or bone tissue such that thetherapeutically useful compound is concentrated within the bone or bonetissue.
 2. The method according to claim 1, wherein the therapeuticallyuseful compound is concentrated within the bone or bone tissue by anexternally applied potential difference.
 3. The method according toclaim 1, wherein the therapeutically useful compound is selected fromthe group consisting of antibiotics, antifungal compounds,chemotherapeutic compounds, tissue growth factors, non-steroidalanti-inflammatory agents, such as indomethacin, neuromuscular agentsaffecting calcium and bone metabolism, anti-viral agents,anti-tuberculosis agents, anthelmintic agents, antiseptic agents,vitamins and minerals.
 4. The method according to claim 1, wherein thetherapeutically useful compound forms a salt in solution and ionises toa single positive or negative ion.
 5. The method according to claim 4,wherein the therapeutically useful compound is an antibiotic selectedfrom the group consisting of flucloxacillin, gentamycin, cephalothin,ticarcillin, ciprofloxacin, nenzl-penicillin, cefoperazone, cefuroxime,cephazolin and tobramycin.
 6. The method according to claim 5, whereinthe antibiotic is gentamycin and wherein it is loaded into the bone orbone tissue at a maximum dose of about 200 mg/kg.
 7. The methodaccording to claim 5, wherein the antibiotic is flucloxacillin andwherein it is loaded into the bone or bone tissue at a maximum dose ofabout 80 mg/kg.
 8. The method according to claim 4, wherein thetherapeutically useful compound is an antifungal compound selected fromthe group consisting of miconazole and ketaconazole.
 9. The methodaccording to claim 4, wherein the therapeutically useful compound is achemotherapeutic compound selected from the group consisting of5-fluoro-uracil and vinblastin.
 10. A bone or bone tissue supplementedwith at least a therapeutically useful compound, wherein said compoundis concentrated within the bone or bone tissue according to the methoddefined by claim
 1. 11. The bone or bone tissue according to claim 10,wherein the therapeutically useful compound is concentrated to an amountbetween the minimum concentration required for activity of the compoundin vivo and the maximum concentration that is equal to the safe maximumsingle dose for systemic administration.
 12. The bone or bone tissueaccording to claim 10 or 11, wherein the therapeutically useful compoundis selected from the group consisting of antibiotics, antifungalcompounds, chemotherapeutic compounds, tissue growth factors,non-steroidal anti-inflammatory agents, such as indomethacin,neuromuscular agents affecting calcium and bone metabolism, anti-viralagents, anti-tuberculosis agents, anthelmintic agents, antisepticagents, vitamins and minerals.
 13. The bone or bone tissue according toclaim 10 or 11, wherein the therapeutically useful compound forms a saltin solution and ionises to a single positive or negative ion.
 14. Thebone or bone tissue according to claim 13, wherein the therapeuticallyuseful compound is an antibiotic selected from the group consisting offlucloxacillin, gentamycin, cephalothin, ticarcillin, ciprofloxacin,nenzl-penicillin, cefoperazone, cefuroxime, cephazolin and tobramycin.15. The bone or bone tissue according to claim 14, wherein theantibiotic is gentamycin and wherein it is loaded into the bone or bonetissue at a maximum dose of about 200 mg/kg.
 16. The bone or bone tissueaccording to claim 14, wherein the antibiotic is flucloxacillin andwherein it is loaded into the bone or bone tissue at a maximum dose ofabout 80 mg/kg.
 17. The bone or bone tissue according to claim 14,wherein the therapeutically useful compound is an antifungal compoundselected from the group consisting of miconazole and ketaconazole. 18.The bone or bone tissue according to claim 14, wherein thetherapeutically useful compound is a chemotherapeutic compound selectedfrom the group consisting of 5-fluoro-uracil and vinblastine.